It is well-known that the function of bioactive proteins can often be enhanced by their combination with other substances. When used to catalyze a reaction or to obtain separation, proteins can be immobilized to increase reaction efficiency and simplify the processing. When used as detecting agents, proteins can be labeled to facilitate measurement. When used to complex with or treat biological organisms, proteins can be combined with bioactive agents (hereinafter called "augmentation") to help achieve treatment efficacy.
Methods for immobilizing proteins are desirable because they localize reaction sites and improve economic recovery. Moreover, immobilized proteins are generally less susceptible to the loss of activity due to chemical attack and changes in temperature and pH than are free proteins.
Methods for labeling or augmenting proteins are desirable because they facilitate quantification, localization, specificity and reactivity of the protein. The resulting combinations, moreover, are the resulting combinations and/or powerful tools for clinical analysis and treatment.
Numerous techniques exist for protein immobilization on solid supports. Proteins can be physically adsorbed onto inert supports or can be covalently bound to the support through reaction with bifunctional linker arms. Microencapsulation, gel entrapment and complexation (with ion exchange resins) also can bind and immobilize.
Numerous techniques also exist for binding labels and bioactive agents to proteins. Most of these techniques call for reaction of the label or agent and a functional group of the protein, such as an amino group, which occurs repeatedly throughout the protein. Although some repetitions of such a group are shielded from binding by the conformation of the protein, many others are exposed and available for binding with the labeling group or bioactive agent. The result is a mixture of proteins having labels or bioactive agents attached at various nonspecific sites.
With any of these techniques for immobilizing, labeling or augmenting, several criteria should be met. The first is a correct spatial orientation for optimum reactivity of the proteins. A protein functions best when it is bound in a fashion that orients its active sites away from the support, label or bioactive agent and renders the sites available for functional operation. The second is the exhibition of protein activities and specificities that are at least comparable to those exhibited by the unbound form of the protein. The third, which is especially applicable to immobilization, is the capability for repeated use and for a high packing density. The fourth is the avoidance of attachment of the support, label or agent within or in the vicinity of the active site of the protein. Otherwise, the resulting loss of functional capacity often causes inadequate reactivity and the need to use more protein.
One of the most important protein embodiments being investigated today is the antibody. The need to minimize the attachment of immobilizing supports, labeling groups or bioactive agents within or near the antigen-binding site of an antibody is widely recognized.
One method for such minimization involves binding antigen to the antibody prior to reaction with the labeling group, bioactive agent, or immobilizing support. In this manner, the antigen shields the antibody binding site from reaction. The success of this shielding method, however, is limited. Although a high affinity of the antibody for the antigen exists, the equilibrium between the antibody/antigen complex and the free antibody/antigen enables free antibody to react. This has foreseeable negative consequences. In addition, exposure of the antigen to a labeling group, bioactive agent, or immobilizing support often results in attachment of that material to the antigen.
The known methods for immobilizing, labeling or augmenting any kind of protein fall far short of maintaining the functional capacity of the protein. Protein reactivity is generally lessened. Proper spatial orientation and packing density are often lacking. And, as a result, many attendant economic, toxic, reactive and non-specificity problems occur. Consequently, better and more specific methods for binding labels, supports or bioactive agents to proteins are needed.
It is an object of the invention, therefore, to develop methods for labeling, immobilizing or bioactively augmenting proteins at sites remote from the functionally active site or sites of the protein. It is also an object of the invention to immobilize proteins by covalently binding them to an inert immobilization support. Another object of the invention is to covalently bind a label to a specific site of a protein. Another object is to covalently bind bioactive agents to a specific location on a protein. A further object of the invention is to immobilize proteins so that they have the correct spatial orientation and packing density which will allow unhindered access to the functionally active site.